LOCUS       sequence-325094-        3279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for: ratiometric pHluorin (cytosolic).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3279)
  AUTHORS   Werley CA, Boccardo S, Rigamonti A, Hansson EM, Cohen AE
  TITLE     Multiplexed Optical Sensors in Arrayed Islands of Cells for 
            multimodal recordings of cellular physiology.
  JOURNAL   Nat Commun. 2020 Aug 4;11(1):3881. doi: 10.1038/s41467-020-17607-5.
  PUBMED    32753572
REFERENCE   2  (bases 1 to 3279)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2020-08-4"; volume: "11(1):3881. doi"; pages: " 
            10.1038/s41467-020-17607-5"
COMMENT     Sequence Label: pMOS017E: Entry vector for: ratiometric pHluor...
FEATURES             Location/Qualifiers
     source          1..3279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             672..1388
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Miesenbock et al., 1998)"
                     /label=ratiometric pHluorin
                     /note="responds to a drop in pH with a reversible decrease 
                     in excitation at 395 nm and a corresponding increase in 
                     excitation at 475 nm"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKDDGNILGHKLEYNYNEHLVYIMADKQKNGTKA
                     IFQVHHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLHTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(702..730)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1325..1345
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    complement(1404..1503)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1520..1539)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1521..1539)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1544..1560)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1544..1560)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1662..1681)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1673..2482
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1740..1759)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2629..3217
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3118..3137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctac catgagtaaa ggagaagaac ttttcactgg agttgtccca attcttgttg
      721 aattagatgg tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg
      781 caacatacgg aaaacttacc cttaaattta tttgcactac tggaaaacta cctgttccat
      841 ggccaacact tgtcactact ttctcttatg gtgttcaatg cttttcaaga tacccagatc
      901 atatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaaagaa
      961 ctatattttt caaagatgac gggaactaca agacacgtgc tgaagtcaag tttgaaggtg
     1021 atacccttgt taatagaatc gagttaaaag gtattgattt taaagatgat ggaaacattc
     1081 ttggacacaa attggaatac aactataacg agcacttggt gtacatcatg gcagacaaac
     1141 aaaagaatgg taccaaagct atctttcaag ttcaccacaa cattgaagat ggaggcgttc
     1201 aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag
     1261 acaaccatta cctgcacaca caatctgccc tttcgaaaga tcccaacgaa aagagagacc
     1321 acatggtcct tcttgagttt gtaacagctg ctgggattac acatggcatg gatgaactat
     1381 acaaatgaat tcgatatcaa gctacccagc tttcttgtac aaagttggca ttataagaaa
     1441 gcattgctta tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat
     1501 ttgccatcca gctgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg
     1561 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     1621 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     1681 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     1741 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     1801 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     1861 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     1921 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     1981 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     2041 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     2101 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     2161 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     2221 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     2281 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     2341 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     2401 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     2461 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat
     2521 tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc
     2581 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2641 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2701 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2761 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2821 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2881 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     2941 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3001 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3061 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3241 acggttcctg gccttttgct ggccttttgc tcacatgtt
//
